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A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s−1 toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP+ (vs. only 2.5% relative activity with NAD+). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP+-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

BPKS from Botrytis cinerea is a novel type III polyketide synthase that accepts C(4)-C(18) aliphatic acyl-CoAs and benzoyl-CoA as the starters to form pyrones, resorcylic acids and resorcinols through sequential condensation with malonyl-CoA. The catalytic efficiency (k(cat)/K(m)) of BPKS was 2.8 × 10(5) s(-1) M(-1) for palmitoyl-CoA, the highest ever reported. Substrate docking analyses addressed the unique features of BPKS such as its high activity and high specificity toward long chain acyl-CoAs.

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The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications.

An efficient biocatalytic cell-free system containing L-arabinitol dehydrogenase (LAD) for L-arabinitol oxidation and NADH oxidase (Nox) for cofactor regeneration was successfully constructed and used for L-rare sugar production. The recombinant LAD (HjLAD) from Hypocrea jecorina suffered from NADH inhibition. Applying Nox to the cell-free HjLAD system drove the thermodynamically unfavorable equilibrium from L-arabinitol to L-xylulose, along with the regeneration of the oxidized cofactor NAD + . Response surface methodology was used to optimize the conditions for L-xylulose production. The optimal conditions for biocatalytic production of L-xylulose were found to be 4.9 U/mL HjLAD, 8.2 mM NAD + at pH 8.0, and 30.9 o C; a high conversion rate of 78.4% was achieved under these conditions. We demonstrate a cellfree enzyme coupling system enabling efficient cofactor recycling and providing high yields of L-xylulose. The results indicate that this coupling system provides a new biocatalytic method for L-rare sugar production.

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